For more than 2 decades, epidemiologic studies have been reported on the effect of dietary fat on the risk of prostate cancer. Experimental data indicate that n-3 polyunsaturated fatty acids (PUFAs) inhibit whereas n-6 PUFAs stimulate prostate cancer cell growth. However; the mechanism of PUFA's effect is unknown. Fish oil is a rich source of n-3 polyunsaturated fatty acids. We hypothesize that n-3 PUFAs incorporate into the sn-2 position of phospholipids, and the n-3 PUIA-enriched phospholipids regulate expression of genes resulting in prostate cell growth inhibition. In this project, we will pursue two parallel approaches to investigate the molecular mechanism involved in the growth inhibition of prostate cells by n-3 PUFA. First, we will use a candidate pathway approach, focusing on the phosphoinositide signaling pathway which is affected by fish oil diet, and which plays a central role in regulating prostate cell growth and survival. Secondly, we will perform a general unbiased screening to identify candidate genes and phospholipids regulated by fish oil diet. Our specific aims are: Assess the effect of n-3 and n-6 PUFA on tumor incidence. Pten+";Nkx3.1 " mice will be fed with n-3 PUFA (fish oil)-enriched, n-6 PUFA-enriched and control diet for a period of 6 months. Prostate cancer incidence will be determined in the dorsolateral''prostate; Determine the effect of fish oil diet on the phoshoinositide signaling pathway. Cancer lesions develop in the dorsolateral but not in the ventral gland of Pten+";Nkx3.1-1" mice. The dorsolateral and ventral prostates will be obtained from diet groups. The levels of phosphoinositides will be measured and the structure of phosphoinositides will be determined. Cell proliferation and apoptosis indexes will be analyzed. mRNA and protein levels of P13K, Pten, AKT, PDK and DIP13 as well as AKT and PDK activity will be compared. Normal and tumor prostate cells will be treated with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and their effect on prostate cell growth and apoptosis will be investigated; Characterize DhosDholiDid and gene expression profiles regulated by diet. The dorsolateral and ventral glands will be laser capture microdissected. Phospholipid (PC, PS, PE, and PI) and mRNA expression profiles will be determined by mass spectrometry and DNA microarray, respectively. Phospholipids and genes that are affected by diet will be identified. The novelty of our application is to use a genetically-defined, immune-competent, orthotopic mouse prostate cancer model and composition-defined diet, in conjunction with cell culture system, to study the effect and mechanism of dietary fat on the phosphoinositide signaling pathway in prostate. Furthermore, we take advantage of our strengths in genomics and lipidomics.' For the first time, molecular changes (mRNA and lipid) associated with fish oil diet in the prostate will be comprehensively and systematically analyzed.